ATP purinergic receptor signalling promotes Sca-1+ cell proliferation and migration for vascular remodelling

Aims Vascular resident stem cells expressing stem cell antigen-1 (Sca-1+ cells) promote vascular regeneration and remodelling following injury through migration, proliferation and differentiation. The aim of this study was to examine the contributions of ATP signalling through purinergic receptor type 2 (P2R) isoforms in promoting Sca-1+ cell migration and proliferation after vascular injury and to elucidate the main downstream signalling pathways. Methods and results ATP-evoked changes in isolated Sca-1+ cell migration were examined by transwell assays, proliferation by viable cell counting assays and intracellular Ca2+ signalling by fluorometry, while receptor subtype contributions and downstream signals were examined by pharmacological or genetic inhibition, immunofluorescence, Western blotting and quantitative RT-PCR. These mechanisms were further examined in mice harbouring TdTomato-labelled Sca-1+ cells with and without Sca-1+-targeted P2R knockout following femoral artery guidewire injury. Stimulation with ATP promoted cultured Sca-1+ cell migration, induced intracellular free calcium elevations primarily via P2Y2R stimulation and accelerated proliferation mainly via P2Y6R stimulation. Enhanced migration was inhibited by the ERK blocker PD98059 or P2Y2R-shRNA, while enhanced proliferation was inhibited by the P38 inhibitor SB203580. Femoral artery guidewire injury of the neointima increased the number of TdTomato-labelled Sca-1+ cells, neointimal area and the ratio of neointimal area to media area at 3 weeks post-injury, and all of these responses were reduced by P2Y2R knockdown. Conclusions ATP induces Sca-1+ cell migration through the P2Y2R–Ca2+–ERK signalling pathway, and enhances proliferation through the P2Y6R–P38-MAPK signalling pathway. Both pathways are essential for vascular remodelling following injury. Video Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12964-023-01185-2.

3 artery, it was pushed and pulled back three times, and left for 3 minutes to facilitate intimal injury. A 9-0 non-absorptive suture was placed below the femoral artery for later use. After the guidewire was withdrawn, the artery was quickly ligated, and about 0.4-cm of surgical suture was left in place to facilitate accurate sampling 3 weeks post-surgery. At that time, the target segment of femoral artery was cut and placed into a centrifuge tube prefilled with OCT, frozen in liquid nitrogen and stored at -80°C until analysis. 1.6 Immunofluorescence Sca-1+ cells treated as indicated on sterile glass slides were washed lightly with PBS, fixed with 4% paraformaldehyde (PFA), blocked with BSA and incubated overnight with anti-P2Y2 receptor (extracellular) antibody (1:200, Alomone, Jerusalem, Israel, #APR-102) and anti-P2Y6 receptor (extracellular) Antibody (1:1000, Alomone, #APR-106). The next day, cells were treated with secondary antibody (donkey anti-rabbit IgG (H+L) Highly Cross-Adsorbed or conjugated to Alexa Fluor 488 (1:500, Thermo Fisher). HelixGen anti-fluorescence quenching agent containing DAPI was added dropwise to prevent fluorescence quenching and to stain nuclei. Images were obtained using a confocal microscope (Zeiss-LSM-980, Germany).

Hematoxylin and eosin staining
Frozen tissue sections were prepared at 10-µm thickness using a cryostat, equilibrated to room temperature, fixed with 4% neutral paraformaldehyde for 20-30 min, washed with running water for 30 s, stained dropwise with hematoxylin for 12-15 min and washed again with running water. The sections were differentiated in 1% hydrochloric acid ethanol for 5 s and soaked in tap water for 10 min, followed by addition of eosin stain for 30-40 s and washing with running water. Subsequently, gradient de-coloration was conducted by soaking in 75%, 85%, 95% and absolute ethanol for 2-5 s each. Tissue sections were made transparent by immersion in xylene for 1-2 s, dried and sealed under coverslips with 1 to 2 drops of neutral resin. Cytoplasm, collagen fibres and muscle fibres were distinguished by different shades of red, while nuclei were distinguished by blue staining.

RT-PCR
Total RNA was isolated from Sca-1+ cells treated as indicated using Nucleozol (Macherey-Nagel, Duren, Germany) according to the manufacturer's instructions. After RNA quality detection and concentration determination using a spectrophotometer, total RNA (1 μg) was reverse-transcribed into cDNA using the PrimerScriptTM RT reagent Kit with gDNA Eraser (TaKaRa). Standard PCR was used to detect the receptor subtypes expressed on Sca-1+ cells, while real-time fluorescent quantitative PCR (RT-qPCR) was used to detect changes in receptor expression induced by ATP. The specific primer pairs used for estimation of gene expression levels are presented in Supplementary Table 1.

Transcriptomics
The effect of ATP on gene expression was examined. Sca-1+ cells were cultured in 6-well dishes (10 cm) and evenly divided into control (C) and ATP (A) treatment groups. Group C was cultured in serum-free medium and group A in serum-free medium containing 30 μM ATP for 18 h. Each culture was quickly washed with PBS, de-plated by trypsinisation for 2 min (stopped with 2 mL PBS), harvested and centrifuged (5 min/137g). The supernatant was removed and 1 mL PBS was added to the precipitated cells. Cells were uniformly dispersed and transferred to 1.5 mL EP tubes for centrifugation (5 min/137g). The supernatant was again discarded and pelleted cells frozen in liquid nitrogen for 4-5 seconds, transferred to a liquid nitrogen tank and shipped on dry ice (−80ºC) to Beijing Nuozhiguan Technology for mRNA-seq analysis using the Illumina sequencing platform. After filtering the original data, checking the sequencing error rate and checking the GC content distribution, we obtained clean reads for subsequent analysis of differentially expressed genes (DEGs). No less than 6.7 Gb of clean data were obtained from each sample, and the R2 between biological replicates was greater than 0.973 in all cases. The DEGs between groups were screened using DESeq2 according to criteria 2. Table   Table 1 Specific primer pairs for purinergic P2 receptor genes for (R-PCR) and RT-PCR reagents

Supplemental Figures:
Gene

P-values in F were calculated using the Nonparametric test (Kruskal-Wallis test) (n = 3 independent experiments). Western blot showing the effect of P2Y2R-shRNA adenovirus at MOI = 300 on P2Y2R protein expression by Sca-1+ cells at 72 hours (G and H). Data in H were first tested by Shapiro-Wilk test for normality and then Ordinary one-way ANOVA (Tukey's multiple comparisons test) was performed. A P< 0.05 is considered a statistically significant test, and statistically significant P-values between the two groups are shown on the graph. (n = 3 independent experiments).
Cui et al.